Structure and regulation of DNA condensates by disordered linker histone tails

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Study of the fundamental factors controlling the transcriptional activity of genes underpins the basic biology of all organisms, and the processes can only be properly understood if the physical nature of condensed genomic DNA (chromatin) is well described. Given this, it is perhaps surprising that there is so much left to understand. Two meters of DNA are condensed into each cell nucleus. This is achieved in two stages. The first stage involves wrapping the DNA around protein spools to form "nucleosomes" that resemble beads on a string. The second stage involves the further condensation of this structure into one that is more compact; this stage is less well understood.Biology can be approached from the top down (i.e. looking at cells) or bottom up (i.e. looking at atomic-level molecules); the hope is always that they join up to make a consistent picture, thus overcoming the inherent limitations and validating the model generated from each approach. Both approaches are necessary to fully understand most biological processes.In the case of chromatin, there is currently a discontinuity concerning the second stage of condensation, since the 30 nm fibre predicted to form in the second stage by a bottom-up approach has not been observed by the best new imaging techniques applied to live cells. Instead, the fibre appears more open, flexible, disordered and heterogeneous, self-assembling into large chromatin globules with liquid-like properties. It seems likely that the highly ordered 30 nm fibre may represent an extreme case of condensed and inactive chromatin, and the more transcriptionally-relevant situation may be much more dynamic. This view is resonant with some alternative views that are emerging from different bottom-up approaches by us and others, that pick up on the high level of inherent disorder in the proteins that package DNA, and their ability to concentrate the DNA into dense liquid condensates, in which the dynamics of the fibre are retained. A liquid condensate is a compelling and more plausible means by which chromatin could respond quickly to environmental stimuli.We have developed a model system that allows us to study, at atomic-level resolution, the way DNA-packaging proteins - specifically the 'linker histones' - bind to the DNA, and the conditions under which the protein/DNA complexes phase separate into dense liquid droplets. It also permits thermodynamic measurements. We would like to exploit this system to answer several key questions: exactly how is DNA bound and condensed by linker histones?; does the highly crowded environment of the resulting condensate allow the entry and action of modifying enzymes known to act in vivo, and how do the condensates respond?; how does the intrinsic disorder of the protein/DNA complexes facilitate rapid exchange by and with other chromatin-associated proteins?; what higher-order structures are present in the condensates, if any, and how is their assembly controlled and regulated?; what are the mechanistic similarities/differences in genome condensation between the many linker histone subtypes (there are for example 11 subtly different linker histones in humans that either come and go through the lifetime of a cell, or locate to sperm or egg) and DNA sequences differing in content and modifications, such as those marking the start of genes or those that are 'silenced' by the addition of methyl groups?In summary, the condensation of the genome is a fundamental process that occurs by a variety of mechanisms across the kingdoms of life. By and large, eukaryotes achieve it in two stages. The first is through the formation of nucleosomes, which has been heavily studied and is well understood. The second is its further condensation by linker histones, which is less well understood, and will be addressed by this proposal.